An understanding of the serum responses of both HIV-1-infected individuals and vaccinees is critical for the development of an effective HIV-1 vaccine. Perhaps the most important biological aspect of a serum response is its neutralizing activity, with a close second being a compendium of its antigenic recognition. We will utilize the tools of structural and computational biology to develop probes to assist in the evaluation of the neutralizing activity of sera, and to decipher the HIV-1 elements recognized by both binding and neutralizing antibodies. Further, these probes will be used for the selection and isolation of B-cells so that their antibody gene loci can be sequenced, enabling the in-depth characterization of secreted antibodies. These capabilities are expected to enhance our understanding of how a broadly neutralizing antibody response develops during the course of infection and also of how the humoral immune system targets vulnerable regions on the HIV-1 Env glycoprotein. The tools of computational design allow for the manipulation of both the surface of a protein as well as its interior. Surface manipulation allows for a precise control of antigenicity, whereas interior manipulation allows for physical properties of flexibility and stability to be altered, thereby modulating surface antigenicity. Together these tools should allow for a precise understanding of elicited serum responses. Such an understanding should facilitate the iterative structure-based improvement of immunogens. Next-generation sequencing allows hundreds of thousands of antibody sequences to be obtained directly from patient sera, thus enabling quantitative analysis of serum response, better understanding of antibody maturation process, and identification of broadly neutralizing antibodies. Due to the natural variation of antibody sequences and inherent sequencing errors, novel bioinformatics techniques have to be developed for antibodyome analysis. These techniques will play a critical role in sera analysis and antibody identification. Identified antibodies can represent natural solutions to antibody optimization, which has potential implications for therapeutic and/or passive transfer studies. For example, this year we identified, characterized, and tracked developmental pathways of one antibody lineage against HIV-1 Membrane-Proximal External Region (MPER) from natural-elicited donor and five HIV-1 fusion peptide(FP) directed neutralizing antibody lineages in vaccinated macaques; these revealed how broadly neutralizing antibodies could be elicited. We have also analyzed naturally elicited antibodies against HIV-1 CD4 binding site (CD4bs) and hepatitis B virus core antigen. Thus, our antibodyome analysis involves understanding development of a target lineage to achieve neutralization and how B cell receptors respond to vaccination in the general population.